The widespread exploitation of monoclonal antibodies for medical research activities, therapeutic treatments, and medical diagnostic immunoassays constitutes a multi-billion dollar health care industry. This project will investigate two types of novel synergistic binding reactions recently described for selected antibody-antigen pairs: those where the affinity of the antibody appeared to increase as the antigen concentration increased (positive cooperativity); and those where the affinity of an antibody directed toward one epitope on a protein antigen appeared to increase in the presence of a second antibody directed toward a different, separate epitope on the same protein antigen. The long-term goal of this project is to understand the molecular mechanisms of these novel synergistic binding reactions. Experiments proposed for the current grant period will focus primarily on functional studies using intact antibodies and corresponding Fab fragments derived from proteolytic cleavage of the parent structures. Functional studies will include kinetic exclusion assays on a KinExA flow fluorimeter and competition assays on a BIAcore surface plasmon resonance spectrometer. Specific aim #1 is to conduct detailed binding studies on monoclonal antibodies that exhibit synergistic binding behavior with their respective antigens. One goal of this aim is to determine whether certain antibodies possess more than two antigen binding sites per intact IgG molecule. Specific aim #2 is to conduct binding studies on protein antigens that bind monoclonal antibodies to separate epitopes in a synergistic manner. One goal of this aim is to quantify the relative contributions of both avidity effects and binding-dependent protein conformation changes to the apparent synergy of binding. In both aims, the unifying hypothesis is that the underlying molecular mechanisms are protein conformation changes that occur as a consequence of the antibody-antigen binding interaction. Any attempts to exploit antibodies that exhibit novel synergistic binding properties to enhance current clinical diagnostic or therapeutic applications must be dependent on existing knowledge concerning the molecular mechanisms whereby the novel binding is expressed. This project is expected to contribute fundamental knowledge toward manipulating these unexpected activities for diagnostic and therapeutic applications. It will also contribute to a basic understanding of a heretofore unrecognized aspect of antibody function.